CN107884485A - A kind of method for the monose composition for determining Yupingfeng complex polysaccharide - Google Patents
A kind of method for the monose composition for determining Yupingfeng complex polysaccharide Download PDFInfo
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- CN107884485A CN107884485A CN201710983393.6A CN201710983393A CN107884485A CN 107884485 A CN107884485 A CN 107884485A CN 201710983393 A CN201710983393 A CN 201710983393A CN 107884485 A CN107884485 A CN 107884485A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a kind of method for the monose composition for determining Yupingfeng complex polysaccharide, it comprises the following steps：1) Yupingfeng complex polysaccharide hydrolyzation sample is prepared；2) mixing monose standard items are prepared；3) pre-column derivatization is handled：Mixing monose hydrolysis standard items obtained by Yupingfeng complex polysaccharide hydrolyzation sample obtained by step 1) and step 2) are mixed with 0.5mol/L PMP methanol solutions and 0.3mol/L NaOH solution respectively, shake well, 30min is reacted under 68 DEG C of water bath conditions, after be cooled to room temperature, the hydrochloric acid for adding 0.3mol/L is neutralized, chloroform is added to be extracted, centrifugation, discard organic layer, repeat extraction 2~3 times, obtain upper strata aqueous, through 0.22 μm of filtering with microporous membrane, Yupingfeng complex polysaccharide derivatization treatment solution and mixing monose derivatization treatment solution are obtained respectively, it is standby；4) high-efficient liquid phase chromatogram technique analysis：Determine the monose composition and its content of Yupingfeng complex polysaccharide.High sensitivity of the present invention, amount of samples is few, stability is good, detects the monose composition in Yupingfeng complex polysaccharide with realizing simple to operate and lower cost.
Description
Technical field
The present invention relates to assay method field, the monose for more particularly to determining polysaccharide forms.
Background technology
Herbal polysaccharide has the extensive biological activity such as strengthen immunity, resisting stress, antiviral, is led in animal and veterinary
Domain is widely used, and the focus of novel chiral synthon research.Yupingfeng San comes from first ZHU Dan-xi《Danxi's experiential therapy》, in being
The representative prescription strengthened the body resistance to consolidate the constitution is cured, is made up of the Radix Astragali, the bighead atractylodes rhizome, windproof three kinds of medicines, Yupingfeng complex polysaccharide is Yupingfeng San
Effective part group, research show that Yupingfeng San and its polysaccharide have extensive adjustment effect to immune system, veterinary clinic have compared with
Good application prospect.
The quality control standard imperfection of veterinary Chinese medicine polysaccharide at present, often only detects total sugar content, causes some false, bad
Polyose occur repeatedly.Pharmacological activity possessed by polysaccharide is closely related with its monose composition, therefore total in detection
Also need to form its monose during sugared content and carry out qualitative analysis., can not be direct because polysaccharide and monose all do not have UV absorption
It is measured, therefore establishes a kind of method meaning weight of the monose composition of the Yupingfeng complex polysaccharide of simple efficiently and accurately
Greatly.
The content of the invention
It is an object of the invention to for above-mentioned the deficiencies in the prior art, there is provided a kind of list for determining Yupingfeng complex polysaccharide
The method of sugar composition, which solves polysaccharide and monose does not have UV absorption, the problem of directly can not being measured to it, and should
Assay method is simply efficient, and precision is high, and important basic number is provided for the initiative of the new veterinary formulations of Yupingfeng complex polysaccharide
According to, also for polysaccharide method of quality control research provides reference and reference.
The technical solution used in the present invention is：A kind of method for the monose composition for determining Yupingfeng complex polysaccharide, it is wrapped
Include following steps：
1) Yupingfeng complex polysaccharide hydrolyzation sample is prepared：Take the compound refined polysaccharide of Yupingfeng and 2mol/L trifluoroacetic acid molten
Liquid mixing is neutralized to neutrality, centrifugation goes supernatant to obtain after water bath processing 8.5h at 98 DEG C with 4mol/L sodium hydroxide solution
Yupingfeng complex polysaccharide hydrolyzation sample, it is standby；
2) mixing monose standard items are prepared：Take D-MANNOSE, rhamnose, D-Glucose, D-ribose, D- galacturonic acids,
D- galactolipins and arabinose, which are codissolved in water, must mix monose standard items, standby；
3) pre-column derivatization is handled：Respectively will be mixed obtained by Yupingfeng complex polysaccharide hydrolyzation sample obtained by step 1) and step 2)
Close monose hydrolysis standard items to mix with 0.5mol/L PMP methanol solutions and 0.3mol/L NaOH solution, shake well, 68 DEG C
React 30min under water bath condition, after be cooled to room temperature, the hydrochloric acid for adding 0.3mol/L is neutralized, and is added chloroform and is extracted
Take, centrifuge, discard organic layer, repeat extraction 3 times, obtain upper strata aqueous, through 0.22 μm of filtering with microporous membrane, obtain Yupingfeng respectively and answer
Polysaccharide derivatization treatment solution and mixing monose derivatization treatment solution are closed, it is standby；
4) high-efficient liquid phase chromatogram technique analysis：It is compound to the Yupingfeng obtained by step 3) more respectively as high performance liquid chromatography
The chromatogram of sugared derivatization treatment solution and mixing monose derivatization treatment solution carries out, to when analyzing, determining that Yupingfeng is compound
The monose composition and its content of polysaccharide.
Specifically, the temperature in step 1) hydrolytic process and water-curing treatment duration are particularly important, and the present invention is through having been surprisingly found that
Yupingfeng complex polysaccharide can be fully hydrolyzed at 98 DEG C after water bath processing 8.5h, to improve the accuracy of late detection data.Together
Manage, in step 3) pre-column derivatization processing procedure, will be defined in 68 DEG C, water-curing treatment duration is limited to 30min so that Yupingfeng
Complex polysaccharide derivatization treatment is more abundant.
As the further improvement of such scheme, the compound refined polysaccharide of Yupingfeng described in step 1) is compound for Yupingfeng
Thick many candies are after the concentration of AB-8 macroporous resin purifications through obtained by vacuum freeze drying.
As the further improvement of such scheme, the compound Thick many candies of Yupingfeng described in step 1) is take medicinal Astragalis, white
Art, it is windproof by weight be 3:1:1 mixing be used as material, then by feed liquid weight ratio is 1:12nd, extraction time 1h, extraction temperature 72
DEG C, extraction time 3 times, by filter, concentrated extracting solution, absolute ethyl alcohol precipitation concentration 12h, the vacuum dried institute of taking precipitate
.Specifically, when the present invention obtains solid-liquid ratio, extraction to medicinal Astragalis, the bighead atractylodes rhizome and the windproof extraction compound Thick many candies process of Yupingfeng
Between, the restriction of extraction temperature and extracting times substantially increase extracting efficiency, avoid unnecessary waste of raw materials.
As the further improvement of such scheme, high performance liquid chromatography is by the Yupingfeng complex polysaccharide in step 4)
Derivatization treatment solution and mixing monose derivatization treatment solution are respectively through chromatogram post separation, with phosphate buffer solution and acetonitrile
Solution is 85 by weight：15 mixed liquor is eluted, and controls 40 DEG C, flow velocity 1mL/min of column temperature, the μ L of sample size 20 detections
Wavelength 254nm.
The beneficial effects of the invention are as follows：The present invention is by establishing PMP pre-column derivatizations-high performance liquid chromatography to Yupingfeng
The monosaccharide component and content of complex polysaccharide are analyzed, the method that its high sensitivity, amount of samples are few, stability is good, of the invention
Accurately and reliably, each monosaccharide component can be separated well, and it is compound more to detect Yupingfeng with realizing simple to operate and lower cost
Monose composition in sugar.
Brief description of the drawings
Fig. 1 is the high-efficient liquid phase analysis chromatogram that monose derivatization treatment solution is mixed in embodiment 1 in the present invention；
Fig. 2 is the high-efficient liquid phase analysis chromatogram of Yupingfeng complex polysaccharide derivatization treatment solution in embodiment 1 in the present invention
Figure.
Embodiment
The present invention is specifically described with reference to embodiment, in order to art personnel to the present invention
Understand.It is necessary that herein the present invention will be further described it is emphasized that embodiment is only intended to, it is impossible to be interpreted as to this
The limitation of invention protection domain, art person skilled in the art, the non-intrinsically safe made according to foregoing invention content to the present invention
The modifications and adaptations of property, should still fall within protection scope of the present invention.Simultaneously following mentioned raw materials are unspecified, are
Commercially available prod；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art or
Preparation method.
Embodiment
A kind of method for the monose composition for determining Yupingfeng complex polysaccharide, it comprises the following steps：
1) the compound Thick many candies of Yupingfeng are prepared：Take medicinal Astragalis, the bighead atractylodes rhizome, it is windproof by weight be 3:1:1 mixing is used as thing
Material, then by feed liquid weight ratio be 1:12nd, extraction time 1h, 72 DEG C of extraction temperature, extraction time 3 times, by suction filtration, concentration extraction
Liquid, absolute ethyl alcohol precipitation concentration 12h, taking precipitate is vacuum dried, obtains the compound Thick many candies of Yupingfeng；
2) the compound refined polysaccharide of Yupingfeng is prepared：Take the Yupingfeng of gained in step 1) compound refined through AB-8 macroreticular resins
Through vacuum freeze drying after purifying concentration, the compound refined polysaccharide of Yupingfeng is obtained；
3) Yupingfeng complex polysaccharide hydrolyzation sample is prepared：Take the compound refined polysaccharide 50mg and 2mol/L of Yupingfeng trifluoro second
Acid solution 2mL mixing is neutralized to neutrality, centrifugation is gone after water bath processing 8.5h at 98 DEG C with 4mol/L sodium hydroxide solution
Clear liquid obtains Yupingfeng complex polysaccharide hydrolyzation sample, standby；
4) mixing monose standard items are prepared：Take D-MANNOSE, rhamnose, D-Glucose, D-ribose, D- galacturonic acids,
D- galactolipins and each 5mg of arabinose, which are codissolved in water, must mix monose standard items, standby；
5) pre-column derivatization is handled：Yupingfeng complex polysaccharide hydrolyzation sample obtained by 100 μ L steps 1) and 100 μ L are walked respectively
Rapid 2) gained mixing monose hydrolysis standard items and 50 μ L 0.5mol/L PMP methanol solutions and 50 μ L 0.3mol/L NaOH are molten
Liquid mixes, shake well, reacts 30min under 68 DEG C of water bath conditions, after be cooled to room temperature, add 50 μ L 0.3mol/L hydrochloric acid
Neutralized, add 1mL chloroforms and extracted, centrifuged, discard organic layer, repeated extraction 3 times, upper strata aqueous is obtained, through 0.22 μ
M filtering with microporous membrane, Yupingfeng complex polysaccharide derivatization treatment solution and mixing monose derivatization treatment solution are obtained respectively, it is standby
With；
6) high-efficient liquid phase chromatogram technique analysis：The Yupingfeng complex polysaccharide derivatization treatment solution and mixing monose are derived
Change processing solution respectively through Wondasil C18 (250mm × 4.6mm, 5 μm) chromatogram post separation, with phosphate buffer solution and second
Nitrile solution is 85 by weight：15 mixed liquor is eluted, and controls 40 DEG C, flow velocity 1mL/min of column temperature, the μ L of sample size 20 inspections
Wavelength 254nm is surveyed, each chromatographic peak obtains good separation after sample introduction measure, wherein mixing monose derivatization treatment solution is efficient
Liquid phase analysis chromatogram such as accompanying drawing 1, the high-efficient liquid phase analysis chromatogram such as accompanying drawing of Yupingfeng complex polysaccharide derivatization treatment solution
2.Mixing monose derivatization treatment solution is distinguished into the μ L of sample introduction 1,2,4,6,8,10, the mass concentration of each monose returned with peak area
Return, linear result and equation of linear regression are as shown in table 1 below.Take Yupingfeng complex polysaccharide derivatization treatment in replica test molten
Each monose peak area result of the continuous 6 sample introductions measure of liquid, obtains average value, substitutes into the equation of linear regression of each monose and obtain
Individual monose mol ratio, it is as a result as shown in table 2 below, be calculated D-MANNOSE in Yupingfeng complex polysaccharide, D-ribose, rhamnose,
D- galacturonic acids, D-Glucose, D- galactolipins, arabinose material amount ratio be 4.64：1：5.88：2.40：120：
42.9：69.5.
The monose calibration curve equation of table 1
The amount of the material of each monose compares result of calculation in the Yupingfeng complex polysaccharide of table 2
Above-described embodiment is the preferred embodiments of the present invention, all with similar technique of the invention and the equivalence changes made,
The protection category of the present invention all should be belonged to.
Claims (4)
- A kind of 1. method for the monose composition for determining Yupingfeng complex polysaccharide, it is characterised in that comprise the following steps：1) Yupingfeng complex polysaccharide hydrolyzation sample is prepared：The compound refined polysaccharide of Yupingfeng and 2mol/L trifluoroacetic acid solution is taken to mix Close after water bath processing 8.5h at 98 DEG C, be neutralized to neutrality with 4mol/L sodium hydroxide solution, centrifugation goes supernatant to obtain jade screen Wind complex polysaccharide hydrolyzation sample, it is standby；2) mixing monose standard items are prepared：Take D-MANNOSE, rhamnose, D-Glucose, D-ribose, D- galacturonic acids, D- half Lactose and arabinose, which are codissolved in water, must mix monose standard items, standby；3) pre-column derivatization is handled：It is respectively that mixing obtained by Yupingfeng complex polysaccharide hydrolyzation sample obtained by step 1) and step 2) is single Syrup solution standard items mix with 0.5mol/L PMP methanol solutions and 0.3mol/L NaOH solution, shake well, 68 DEG C of water-baths Under the conditions of react 30min, after be cooled to room temperature, the hydrochloric acid for adding 0.3mol/L is neutralized, and is added chloroform and is extracted, from The heart, organic layer is discarded, repeat extraction 3 times, obtain upper strata aqueous, through 0.22 μm of filtering with microporous membrane, it is compound more to obtain Yupingfeng respectively Sugared derivatization treatment solution and mixing monose derivatization treatment solution, it is standby；4) high-efficient liquid phase chromatogram technique analysis：The Yupingfeng complex polysaccharide obtained by step 3) is spread out respectively as high performance liquid chromatography The chromatogram of biochemical treatment solution and mixing monose derivatization treatment solution carries out, to when analyzing, determining Yupingfeng complex polysaccharide Monose composition and its content.
- A kind of 2. method of monose composition for determining Yupingfeng complex polysaccharide according to claim 1, it is characterised in that：Step It is rapid 1) described in the compound refined polysaccharide of Yupingfeng for the compound Thick many candies of Yupingfeng after the concentration of AB-8 macroporous resin purifications through true Obtained by vacuum freecing-dry.
- A kind of 3. method of monose composition for determining Yupingfeng complex polysaccharide according to claim 2, it is characterised in that：Step It is rapid 1) described in the compound Thick many candies of Yupingfeng for take medicinal Astragalis, the bighead atractylodes rhizome, it is windproof be 3 by weight:1:1 mixing is used as material, It is again 1 by feed liquid weight ratio:12nd, extraction time 1h, 72 DEG C of extraction temperature, extraction time 3 times, by filter, concentrated extracting solution, Absolute ethyl alcohol precipitation concentration 12h, the vacuum dried gained of taking precipitate.
- A kind of 4. method of monose composition for determining Yupingfeng complex polysaccharide according to claim 1, it is characterised in that：Step It is rapid 4) in high performance liquid chromatography be by the Yupingfeng complex polysaccharide derivatization treatment solution and mixing monose derivatization treatment Solution is respectively 85 by weight with phosphate buffer solution and acetonitrile solution through chromatogram post separation：15 mixed liquor is carried out Elution, control 40 DEG C, flow velocity 1mL/min of column temperature, the μ L Detection wavelengths 254nm of sample size 20.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109060668A (en) * | 2018-06-05 | 2018-12-21 | 朱蒙 | Gold paste jade material standard sample |
| CN110453017A (en) * | 2019-07-29 | 2019-11-15 | 佛山科学技术学院 | A kind of method for hydrolysis of Yupingfeng complex polysaccharide |
| CN110658272A (en) * | 2019-09-23 | 2020-01-07 | 佛山科学技术学院 | Method for detecting encapsulation rate of composite polysaccharide liposome |
| CN111965271A (en) * | 2020-07-17 | 2020-11-20 | 佛山科学技术学院 | Quantitative method for polysaccharide absorption |
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2017
- 2017-10-19 CN CN201710983393.6A patent/CN107884485A/en active Pending
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109060668A (en) * | 2018-06-05 | 2018-12-21 | 朱蒙 | Gold paste jade material standard sample |
| CN110453017A (en) * | 2019-07-29 | 2019-11-15 | 佛山科学技术学院 | A kind of method for hydrolysis of Yupingfeng complex polysaccharide |
| CN110658272A (en) * | 2019-09-23 | 2020-01-07 | 佛山科学技术学院 | Method for detecting encapsulation rate of composite polysaccharide liposome |
| CN111965271A (en) * | 2020-07-17 | 2020-11-20 | 佛山科学技术学院 | Quantitative method for polysaccharide absorption |
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Application publication date: 20180406 |